The use of proteins, such as enzymes, as catalysts in industrial-scale synthesis of specialty chemicals and pharmaceuticals has received much attention in recent years [K. Faber and M. C. R. Franssen, Trends in Biochem. Tech., 11, pp. 461-70 (1993)]. Enzymes are recognized as useful tools for accomplishing chemical reactions in a stereo-, regio- and chemoselective manner. The ability of enzymes to function under mild conditions, ease of disposal and minimal waste production are further advantages associated with their use. Enzymes are also used for catalysis in organic solvents to solubilize substrates and products and to manipulate reaction kinetics and equilibrium in order to increase product yield.
While enzymes offer impressive synthetic potential over current nonenzymatic technology, their commercial use has been limited by disadvantages such as poor stability, variability in performance, difficulty of isolation and purification, difficulty in handling, high cost and long reaction times. Furthermore, organic solvents are often incompatible with enzymes, leading to enzyme degradation or inactivation [A. M. Klibanov, "Asymmetric transformations Catalyzed by Enzymes in Organic Solvents", Acc. Chem. Res., 23, pp. 114-20 (1990]. In order for enzymes to function as viable industrial catalysts, they must be able to function without excessive intervention in the practical environments of manufacturing processes. Such environments include polar and non-polar organic solvents and aqueous-organic solvent mixtures. The low activity of enzymes and their aversion to organic solvents have remained barriers to widespread use of these proteins in routine organic syntheses. Even when such syntheses are catalyzed by enzymes, it is not unusual to see processes employing more enzyme than substrate by weight. See, for example, R. Bovara et al., Tetrahedron: Asymmetry, 2, pp. 931-38 (1991); Y. -F. Wang et al., J. Am. Chem. Soc., 110, pp. 7200-05 (1988); A. Palomaer et al., Chirality, 5, pp. 320-28 (1993) and V. Gotor et al., Tetrahedron, 47, pp. 9207-14 (1991).
Two methods designed to overcome these disadvantages--enzyme purification and enzyme immobilization--have addressed some of these disadvantages. However, they have not solved the problem of loss of enzyme activity or stability in organic solvents. Immobilization has actually exacerbated these problems by incurring higher costs and diluting the activity of the enzyme by the addition of support materials. Enzyme purification also incurs added cost and, in most cases fails to increase enzyme activity in organic solvents. For example, the potential synthetic benefits of purified lipases in organic solvents have not been realized [T. Nishio and M. Kamimura, Agric. Biol, Chem., 52, pp. 2631-32 (1988); T. Yamane et al., Biotechnol. Bioeng., 36, pp. 1063-69 (1990)]. The cost of purified lipases is higher than that of their crude counterparts, while their stability and activity in organic solvents is lower [R. Bovara et al., Biotechnol. Lett., 15, pp. 169-74 (1993); E. Wehtje et al., Biotechnol. Bioeng., 41, pp. 171-78 (1993); G. Ottolina et al., Biotechnol. Lett., 14, pp. 947-52 (1992)].
Recent studies have demonstrated that enzyme activity in organic solvents is intimately related to water content, size and morphology of the catalyst particles and the enzyme microenvironment [A. M. Klibanov, "Enzymatic Catalysis in Anhydrous Organic Solvents", Trends in Biochem. Sci., 14, pp. 141-44 (1989)]. These parameters have been adjusted by preparing lyophilized complexes of enzymes with carbohydrates, organic buffers or salts [K. Dabulis and A. M. Klibanov, Biotechnol. Bioeng., 41, pp. 566-71 (1993); A. D. Blackwood et al., Biochim. Biophys. Acta, 1206, pp. 161-65 (1994); Y. L. Khmelnitsky et al., J. Am. Chem. Soc., 116, pp. 2647-48 (1994)]. However, despite the widespread use of lyophilization for preparation of enzymes for catalysis in organic solvents, its impact is not fully understood and, in some instances, it may cause significant reversible denaturation of enzymes [Dabulis and Klibanov, supra].
Other approaches to the problem of low enzyme activity in biotransformations involving organic solvents have included the use of surfactants. It has been reported that non-ionic surfactants added prior to immobilization of lipase into a photo-crosslinkable resin pre-polymer, or added to the reaction incubation mixture, increase enzyme activity [B. Nordvi and H. Holmsen, "Effect of Polyhydroxy Compounds on the activity of Lipase from Rhizopus arrhizus in Organic Solvent", in Biocatalysis in Non-Conventional Media, J. Tramper et al. (Ed.), pp. 355-61 (1992)]. Immobilized lipase enzymes prepared by application of a non-ionic surfactant (containing at least one fatty acid moiety) to a hydrophobic support prior to or simultaneously with application of the lipase enzyme demonstrate activity in enzymatic conversion processes comparable with conventional immobilized enzymes [PCT patent application WO 94/28118].
Surfactants have been said to reduce enzymatic activity of lipases [S. Bornemann et al., "The Effects of Surfactants on Lipase-Catalysed Hydrolysis of Esters: Activities and Stereo Selectivity", Biocatalysis, 11, pp. 191-221 (1994)]. Nevertheless, surfactants have been mixed with an aqueous solution of an enzyme, the mixture dewatered and the resulting enzyme preparation used as a catalyst said to have enhanced activity in organic solvents [PCT patent application WO 95/17504]. Surfactants or lipids have also been used to coat enzymes in order to solubilize them in organic solvents and, thus, increase chemical reaction rates [M. Goto et al., "Design of Surfactants Suitable for Surfactant-Coated Enzymes as Catalysts in Organic Media", J. Chem. Eng. Jpn., 26, pp. 109-11 (1993); N. Kamiya et al., "Surfactant-Coated Lipase Suitable for the Enzymatic Resolution of Methanol as a Biocatalyst in Organic Media", Biotechnol, Prog., 11, pp. 270-75 (1995)]. After this procedure, the enzymes become soluble in organic solvents. Enzyme complexes soluble in organics are also described in V. M. Paradkar and J. S. Dordick, J. Am. Chem. Soc., 116, pp. 5009-10 (1994) (proteases) and Y. Okahata et al., J. Org. Chem., 60, pp. 2240-50 (1995)(lipases).
The advent of crosslinked enzyme crystal ("CLEC.TM.") technology provided a unique approach to solving the above-described disadvantages [N. L. St. Clair and M. A. Navia, J. Am. Chem. Soc., 114, pp. 7314-16 (1992)]. Crosslinked enzyme crystals retain their activity in environments that are normally incompatible with enzyme (soluble or immobilized) function. Such environments include prolonged exposure to high temperature and extreme pH. Additionally, in organic solvents and aqueous-organic solvent mixtures, crosslinked enzyme crystals exhibit both stability and activity far beyond that of their soluble or conventionally-immobilized counterparts. Since so many biocatalysis processes depend on stability and activity of an enzyme under sub-optimal conditions, crosslinked enzyme crystals are advantageously used in industrial, clinical and research settings enzymes. Thus, crosslinked enzyme crystals represent an important advance in the area of biocatalysis, as attractive and broadly applicable catalysts for organic synthesis reactions [R. A. Persichetti et al., "Cross-Linked Enzyme Crystals (CLECs) of Thermolysin in the Synthesis of Peptides", J. Am. Chem. Soc., 117, pp. 2732-37 (1995) and J. J. Lalonde et al., J. Am. Chem. Soc., 1117, pp. 6845-52 (1995)].
Despite the progress of protein catalysis technology in general, the need still exists for catalysts which have high activity in organic solvents.